Hello, I do the HPRT assay as well, though not the BrdU. Is that the
quick one with autoradiography?
My pharmacology book says that it is incorporation into DNA that kills the
cells, and that it is a delayed sort of toxicity. 6-TG incorporation does
not "cap" the DNA and prevent replication, but its incorporation somehow
leads to strand breaks. The toxicity iis "delayed", cells do not die in
one generation of exposure. That is why you probably have to use such
high concentrations in the BrdU assay, to really shut down normal HPRT
cells so that the BrdU incorporation truly goes into the mutants. And
since you do not have to recover the mutants as clones, it is fine to put
extremely high concentrations of 6-TG into the culture if you don't expect
to recover clones.
Joe Wiemels
Sabine Hanelt (sabine.hanelt at medizin.uni-ulm.de) wrote:
: Hi netters,
: in our group we are working with the hprt mutation assay and the BrdU assay to
: detect HPRT deficient clones.
: Since some weeks we have a never ending discussion about two things.
: The first is: what does 6-TG (thioguanine) exactly do so that hprt proficient
: cells die. Does the polymerization stop or are the enzymes not able to
: translate mRNA with 6-TG into functional proteins, or both of it.
: The second is: for the BrdU assay we use 6-TG in much higher concentrations
: (34 µg/ml) than used in the "classical" hprt assay (2-10 µg/ml). Does 6-TG
: bloc the growth of the cells when it is used in higher concentrations and if
: this is the case, in which form does it bloc.
: Thank's for your help
: Sabine